In the bleeding individual, coagulation is initiated by the Tissue Factor/Factor VIIa (TF/FVIIa) complex when extravascular TF is exposed to FVIIIa in the blood. TF/FVIIa complex formation leads to the activation of Factor X (FX) to FXa which, together with activated Factor V (FVa), generates a limited amount of thrombin. Small amounts of thrombin activate platelets which, in turn, results in the surface exposure of platelet phospholipids that supports the assembly and binding of the tenase complex composed of activated Factor VIII (FVIIIa) and Factor IX (IXa). The tenase complex is a very efficient catalyst of FX activation and FXa generated in this second step serves as the active protease in the FVa/FXa pro-thrombinase complex responsible for the final thrombin burst. Thrombin cleaves fibrinogen to generate fibrin monomers, which polymerise to form a fibrin network which seals the leaking vessel and stops the bleeding. The rapid and extensive thrombin burst is a prerequisite for the formation of a solid and stable fibrin clot.
An inadequate propagation of FXa and thrombin generation caused by FVIII or FIX deficiency is the reason underlying the bleeding diathesis in haemophilia A and B patients, respectively. In people with haemophilia, FXa generation is primarily driven by the TF/FVIIa complex because FVIII or FIX deficiency leads to rudimentary FXa generation by the tenase complex. TF/FVIIa-mediated activation of FX to FXa is, however, temporary because tissue factor pathway inhibitor (TFPI) inhibits Factor Xa and the TF/FVIIa complex in an auto-regulatory loop. Feed-back inhibition leads to formation of a TF/FVIIIa/FXa/TFPI complex. Neutralizing TFPI inhibition prolongs TF/FVIIa-mediated activation of FX during initiation of coagulation, and thereby it promotes haemostasis in people with haemophilia with an inadequate FXa generation caused by impaired tenase activity due to e.g. FVIII or FIX deficiency.
TFPI is a slow tight-binding competitive inhibitor which regulates FX activation and activity through inhibition of both TF-FVIIa and FXa. TFPI inhibition of FXa occurs in a biphasic reaction that initially leads to a loose TFPI-FXa complex which slowly rearranges to a tight binding TFPI-FXa complex where the second Kunitz-type inhibitor domain of TFPI (KPI-2) binds and blocks the active site of FXa. Following initiation of coagulation, TF/FVIIa-mediated FXa generation is tightly down-regulated by TFPI. TF/FVIIa is inhibited by TFPI in a process which as a rate limiting step involves TFPI inhibition of FXa, either when FXa is bound to the TF/FVIIa complex or bound in its near vicinity on the membrane (Baugh et al., 1998, JBC, 273: 4378-4386). The first Kunitz-type inhibitor domain of TFPI (KPI-1) contributes to the formation of the tight TFPI-FXa complex and it directly binds and blocks the active site of TF-bound FVIIIa. The C-terminal part of TFPI, consisting of the third Kunitz-type inhibitor domain, KPI-3, and the basic C-terminal tail, does not have any direct inhibitory activity, but it enhances the formation of the TFPI-FXa complex and it binds to Protein S and to heparin-like molecules which are involved in localising TFPIα to the vascular surface.
TFPIα inhibits FXa-mediated thrombin generation by the prothrombinase complex at physiologically relevant TFPIα concentrations. This feed-back inhibition works primarily as a temporal impediment of the initiation phase of clot formation. TFPIα inhibition of FXa in the prothrombinase complex is mediated via a high-affinity interaction between the basic region of TFPIα and an acidic region in FV, which is exposed on platelet FVa and on FVa when initially activated by FXa. The FVa-dependent inhibitory activity of TFPIα is lost upon removal of the acidic region when FVa is further cleaved by thrombin generated by the prothrombinase complex. TFPIβ lacks the C-terminal basic region and is therefore not an inhibitor of prothrombinase activity (Wood et al. 2013, PNAS, 110: 17838-843).
Antibodies that are capable of binding TFPI are known in the art. For example, WO2010/072691, WO2012/001087 and WO2012/135671 disclose monoclonal antibodies (mAbs), each of which is capable of binding to one specific epitope of TFPI. The following limitations may apply to such an antibody that targets a single TFPI epitope, e.g. on a KPI domain, typically restricted to the paratope area of an antibody defined by a single variable region. First of all, the final inhibition of the TF/FVIIa/FXa complex is dependent on several interactions between complementary areas scattered over TFPI and the TF/FVIIa/FXa complex. This applies not only to the direct binding of KPI-1 and KPI-2 of TFPI to the active sites of FVIIa and FXa, respectively, but also to interactions with TF/FVIIa/FXa exosites which involve regions of the N- and C-terminal regions of TFPI. A monoclonal antibody that binds, for example, a single KPI may not be capable of completely blocking all inhibitory functions of TFPI, particularly at physiologically elevated concentrations of TFPI. Secondly, targeting TFPI with a monoclonal antibody or fragment thereof may cause TFPI to accumulate in the circulation as a result of a reduced renal clearance of the TFPI-mAb complex, or as a result of other clearance mechanisms which are reduced due to TFPI-mAb complex formation. Dosing of some monoclonal antibodies may also cause release of TFPI from the endothelium and rapidly increasing plasma TFPI levels, similarly to what has been observed after dosing of heparin or an aptamer, which binds to KPI-3 and the C-terminal tail of TFPI (Wong et al. Haemophilia, 2012, 18: LB-WE 03.1 p 831, Hoppensteadt et al., Thromb. Res., 77: 175-185). Thirdly, it may be desirable to target a specific pool of TFPI. Full length circulating TFPIα is thought to be of particular importance for the regulation of coagulation at a site of injury. The fact that, of all TFPI pools, only full-length TFPIα possesses an exposed C-terminal region (residues 182-276) makes it possible to selectively target the full-length TFPIα pool. By only targeting the full-length TFPIα pool and not, for example, TFPIβ or lipoprotein associated TFPI, target mediated drug disposition may be reduced, leading to prolonged in vivo drug half-lives and lower dose requirements. However, known antibodies specifically targeting the C-terminal region of TFPIα are not capable of completely neutralising TFPIα activity, especially at elevated concentrations of TFPI. The inventors envisage that the antibodies—and combinations thereof—that are disclosed herein may address such limitations.